This invention relates generally to plant genetic engineering, and specifically to novel genetically engineered plants characterized as having a phenotype of modulated flower development and methods for producing such plants.
Most angiosperm species are induced to flower in response to environmental stimuli such as day length and temperature, and internal cues, such as age. Adult organs of flowering plants develop from groups of stem cells called meristems. The identity of a meristem is inferred from structures it produces: vegetative meristems give rise to roots and leaves, inflorescence meristems give rise to flower meristems, and flower meristems give rise to floral organs such as sepals and petals. Not only are meristems capable of generating new meristems of different identity, but their own identity can change during development. For example, a vegetative shoot meristem can be transformed into an inflorescence meristem upon floral induction, and in some species, the inflorescence meristem itself will eventually become a flower meristem. Despite the importance of meristem transitions in plant development, little is known about the underlying mechanisms.
Following germination, the shoot meristem produces a series of leaf meristems on its flanks. However, once floral induction has occurred, the shoot meristem switches to the production of flower meristems. Flower meristems produce floral organ primordia, which develop individually into sepals, petals, stamens or carpels. Thus, flower formation can be thought of as a series of distinct developmental steps, i.e. floral induction, the formation of flower primordia and the production of flower organs. Mutations disrupting each of the steps have been isolated in a variety of species, suggesting that a genetic hierarchy directs the flowering process (see for review, Weigel and Meyerowitz, In Molecular Basis of Morphogenesis (ed. M. Bernfield). 51st Annual Symposium of the Society for Developmental Biology, pp. 93-107, New York, 1993).
Recently, studies of two distantly related dicotyledons, Arabidopsis thaliana and Antirrhinum majus, led to the identification of three classes of homeotic genes, acting alone or in combination to determine floral organ identity (Bowman, et al., Development, 112:1, 1991; Carpenter and Coen, Genes Devl., 4:1483, 1990; Schwarz-Sommer, et al., Science, 250:931, 1990). Several of these genes are transcription factors whose conserved DNA-binding domain has been designated the MADS box (Schwarz-Sommer, et al., supra).
Earlier acting genes that control the identity of flower meristems have also been characterized. Flower meristems are derived from inflorescence meristems in both Arabidopsis and Antirrhinum. Two factors that control the development of meristematic cells into flowers are known. In Arabidopsis, the factors are the products of the LEAFY gene (Weigel, et al., Cell 69:843, 1992) and the APETALA1 gene (Mandel, et al., Nature 360:273,1992). When either of these genes is inactivated by mutation, structures combining the properties of flowers and inflorescence develop (Weigel, et al., supra; Irish and Sussex, Plant Cell, 2:741, 1990). In Antirrhinum, the homologue of the Arabidopsis LEAFY gene is FLORICAULA (Coen, et al., Cell, 63:1311, 1990) and that of the APETALA1 gene is SQUAMOSA (Huijser, et al., EMBO J., 11:1239, 1992). The latter pair contains MADS box domains.
Flowering plants exhibit one of two types of inflorescence architecture: indeterminate, in which the inflorescence grows indefinitely, or determinate, in which a terminal flower is produced. In two mutants in distantly related species, terminal flower 1 in Arabidopsis and centroradialis in Antirrhinum, inflorescences that are normally indeterminate are converted to a determinate architecture. The Antirrhinum gene CENTRORADIALIS (CEN) and the Arabidopsis gene TERMINAL FLOWER 1 (TFL1) were shown to be homologous, which suggests that a common mechanism underlies indeterminacy in these plants. However, unlike CEN, TFL1 is also expressed during the vegetative phase, where it delays the commitment to inflorescence development and thus affects the timing of the formation of the inflorescence meristem as well as its identity.
There is increasing incentive by those working in the field of plant biotechnology to successfully genetically engineer plants, including the major crop varieties. One genetic modification that would be economically desirable would be to accelerate the flowering time of a plant. Induction of flowering is often the limiting factor for growing crop plants. One of the most important factors controlling induction of flowering is day length, which varies seasonally as well as geographically. There is a need to develop a method for controlling and inducing flowering in plants, regardless of the locale or the environmental conditions, thereby allowing production of crops, at any given time. Since most crop products (e.g., seeds, grains, fruits), are derived from flowers, such a method for controlling flowering would be economically invaluable.
The present invention is based on the discovery of a gene that regulates flowering in plants. The gene is termed xe2x80x9cflowering locus Txe2x80x9d or xe2x80x9cFTxe2x80x9d and functions to modulate flowering time. Overexpression of FT results in dramatic early flowering in Arabidopsis while loss of function mutations in FT or antisense directed to FT causes late flowering.
In a first embodiment, the invention provides FT polypeptide, which is characterized as having a molecular weight of approximately 20 kD, as determined by SDS-PAGE; being located on chromosome 1 of Arabidopsis; and functioning to modulate flowering time. An exemplary amino acid sequence of FT polypeptide is shown in SEQ ID NO:2. An exemplary FT peptide having flowering promoting activity is shown in SEQ ID NO:4 and more specifically in SEQ ID NO:6. Also included in the invention is an isolated polynucleotide that encodes FT polypeptide. An exemplary nucleotide sequence encoding FT is shown in SEQ ID NO:1.
In another embodiment, the invention provides a genetically modified plant including at least one exogenous nucleic acid sequence such as at least FT-encoding nucleic acid sequence in its genome and characterized as having modulated flower development. Flower development can be inhibited or accelerated by the method of the invention.
In another embodiment, the invention provides a method for genetically modifying a plant cell such that a plant, produced from the cell, is characterized as having modulated flower development as compared with a wild-type plant. The method includes introducing at least FT encoding polynucleotide of the invention into a plant cell to obtain a transformed plant cell; and growing the transformed plant cell under conditions which permit expression of FT polypeptide, thereby producing a plant having modulated flower development.
In yet another embodiment, the invention provides a method of producing a genetically modified plant characterized as having early flower development. The method includes contacting a plant cell with a vector containing a nucleic acid sequence comprising at least a structural gene encoding FT polypeptide, the gene operably associated with a promoter, to obtain a transformed plant cell; producing a plant from the transformed plant cells; and selecting a plant exhibiting early flower development.
In yet another embodiment, the invention provides a method for modulating flower development in a plant cell. The method includes contacting the plant cell with a vector containing a nucleic acid sequence having at least one structural gene encoding FT polypeptide to modulate flower development, operably associated with a promoter to obtain a transformed plant cell; growing the transformed plant cell under plant forming conditions; and inducing early flower development in the plant under conditions and for a time sufficient to modulate flower development. Modulation of flower development includes acceleration or inhibition of development.
The invention also provides a genetically modified plant having a transgene disrupting or interfering with, expression of flowering time gene (FT), chromosomally integrated into the genome of the plant. The invention also includes a method for producing such plants, characterized as having late flower development. The method includes contacting a plant cell with a vector containing a nucleic acid sequence including at least a structural gene disrupting or interfering with expression of FT polypeptide, wherein the gene is operably associated with a promoter, to obtain a transformed plant cell; producing a plant from the transformed plant cells; and selecting a plant exhibiting late flower development. The method also includes substituting the structural gene disrupting or interfering with expression of FT polypeptide with a vector containing an FT antisense nucleic acid sequence or FT dominant-negative encoding nucleic acid sequence.
In another embodiment, the invention provides a method for identifying a compound which modulates FT activity or gene expression. The method includes incubating components including the compound and FT polypeptide or a recombinant cell expressing FT, under conditions sufficient to allow the components to interact; and determining the effect of the compound on the activity or expression of FT. A compound may inhibit or may stimulate the activity or expression of FT.
In another embodiment, the invention provides a method for identifying a peptide of FT that either mimics or inhibits the activity or expression of wild-type FT. Such peptide can be used in place of wild-type FT, wherein wild-type FT is a control, in order to determine the effect on flowering time for the peptide.